IL-17C Reporter HEK 293 Cells

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IL-17C Reporter Cells

Signaling pathway in HEK-Blue™ IL-17C cells

HEK-Blue™ IL-17C cells were engineered from the human embryonic kidney HEK293 cell line to detect bioactive human and murine interleukin-17C (IL-17C) by monitoring the activation of the NF-κB and AP-1 pathways. In addition, these cells can be used for screeningantibodies or small molecule inhibitors targeting the IL-17 pathway.

IL-17C is a pro‑inflammatory cytokine that promotes Th17 immunity to pathogens in the lungs, skin, and colon [1].

 More details

Cell line description

HEK-Blue™ IL-17C cells were generated by stable transfection with the genes encoding for the human IL-17C receptor (IL-17RA and IL-17RE chains), the ACT1 adaptor molecule, as well as an NF-κB- and AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of IL-17C to its receptor triggers a signaling cascade leading to the activation of NF-κB/AP-1 and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue™ IL-17C cells detect human (h) IL-17C and murine (m) IL-17C. Of note they also respond to h/mIL-17E (also known as IL-25), as well as hIL-17A. They display little to no response to hIL-17F and do not respond to mIL-17A nor mIL-17F (see figures). HEK-Blue™ IL-17C cells do not respond to hIL-17B nor hIL-17D (data not shown).

Key features

• Fully functional IL-17 signaling pathway
• Readily assessable NF-κB/AP-1-SEAP reporter activity
• Strong response to h/mIL-17C & h/mIL-17E
• Moderate response to hIL-17A
• Low to no response to mIL-17A & h/mIL-17F

Applications

• Detection and quantification of h/mIL-17C activity
• Detection and quantification of h/mIL-17E (IL-25) activity
• Screening of anti-IL-17 or anti-IL-17 receptor antibodies
• Screening and release assay of small molecule inhibitors of the IL-17 pathway

Reference:

1. Yamaguchi S. et al., 2018. The roles of IL-17C in T cell-dependent and -independent inflammatory diseases. Sci Rep. 8(1):15750.

Figures

Dose-response of HEK-Blue™ IL-17C cells to recombinant IL-17 cytokines.
Cells were stimulated with increasing concentrations of recombinant human or murine IL-17C, IL-17E, and IL-17A. After overnight incubation, the NF-κB response was determined using QUANTI‑Blue™, a SEAP detection reagent, and reading the optical density (OD) at 630 nm. EC50 values are shown as mean ± SD.

Cytokine response profile of HEK-Blue™ IL-17C cells. 
Cells were stimulated with various human and murine recombinant cytokines: 10 ng/ml of hIL‑17C, mIL‑17C, hIL‑17E, mIL‑17E, hIL‑17A, mIL-17A, 1x104 IU/ml of hIFN-α, hIFN-β, 100 ng/ml of hIFN-γ, and 1 ng/ml of hIL-1 β and hTNF-α. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™. Data is shown as mean ± SD.

Dose-dependent inhibition of human IL-17C response using a neutralizing antibody against the human IL-17RA receptor in HEK‑Blue™ IL-17C cells. 
The antibody was incubated with HEK-Blue™ IL-17C cells for 30 minutes prior to the addition of 30 ng/ml of hIL-17C. After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI‑Blue™. Data represent % inhibition of reporter activity.

Specifications

Antibiotic resistance: Blasticidin, Hygromycin, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2-4 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin®

Specificity: human and mouse IL-17C & IL-17E (IL-25), and human IL-17A 

Detection range:

• human & mouse IL-17C: 3 - 100 ng/ml
• human & mouse IL-17E (IL-25): 0.3 - 100 ng/ml
• human IL-17A: 10 - 100 ng/ml

Quality Control:

• SEAP reporter activity in response to IL-17 is validated using functional assays.
• The stability for 20 passages following thawing is confirmed.
• These cells are tested for mycoplasma contamination. 

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 2 x 1 ml of HEK-Blue Selection (250X concentrate)
• 1 ml of Normocin® (50 mg/ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent).

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Details

Interleukin 17 (IL-17) is a family of six closely related cytokines (IL-17A to IL-17F) which can have both pro-inflammatory and anti-inflammatory activities. IL-17C is a pro‑inflammatory cytokine that promotes Th17 immunity to pathogens in the lungs, skin, and colon [1].

IL-17 cytokines exert their biological activities following binding to heterodimeric receptors containing the ubiquitous IL-17RA chain and a second IL-17R (C, B or E) chain. IL-17A and IL-17F bind to the IL-17RA/IL-17RC receptor, IL-17C binds to the IL-17RA/IL-17RE receptor, and IL-17E binds to the IL-17RA/IL-17RB receptor [1, 2]. The activated heterodimeric receptor recruits the Act1 adaptor and induces the TNF receptor-associated factor 6 (TRAF6) ubiquitylation. This triggers a signaling cascade that results in NF-κB and AP-1 activation [3].

1. Yamaguchi S. et al., 2018. The roles of IL-17C in T cell-dependent and -independent inflammatory diseases. Sci Rep. 8(1):15750.
2. Gu C. et al., 2013. IL-17 family: cytokines, receptors and signaling. Cytokine. 64(2):477-85.
3. Pappu R. et al., 2011. The interleukin-17 cytokine family: critical players in host defence and inflammatory diseases. Immunology. 134(1): 8–16.

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